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Disrupting Disease

December 12, 2018

As predators go, the tsetse fly is small and its deadly microscopic trypanosome parasites even smaller.

African sleeping sickness, or trypanosomiasis, is spread by the painful bite of this stocky fly, which inhabits large swaths of rural Africa. Hundreds of East African and thousands of West African trypanosomiasis cases are reported each year. Closely related parasites also ravage herds of domestic cattle.

Serap Aksoy, Ph.D., a professor of epidemiology, is among a small band of global researchers who are unlocking surprising secrets of this neglected disease—and spotting new ways to interrupt it. Together with colleagues in Uganda and Kenya, Aksoy’s team at the Yale School of Public Health has made seminal discoveries about the fly, its bacterial microbiota and trypanosomes, yielding a clever vector control strategy called paratransgenesis that could halt the parasite in its tracks.

The goal, she said, is to “make a fly that is very resistant to the trypanosome’s transmission.” If paratransgenesis works, a tsetse fly bite could one day be more nuisance than death sentence.

The stakes are high, and so is the need for creative new approaches. Though public health officials have long actively tracked down and treated infected people, reducing the number of cases in recent years, sleeping sickness remains a serious threat. Diagnosis can be difficult, with

early symptoms as vague as headache and malaise, and the later stage—with its trademark daytime drowsiness—hard to treat. Chemotherapeutic treatments are decades old and dangerous in their own right, but the risk is necessary, as the disease otherwise kills most people within a few months to years.

Although a successfully treated human can no longer transmit the parasite to a fly, the trypanosome can still hide out in wild and domestic animal reservoirs. Killing the fly with traps and pesticides is largely impractical; it ranges over 10 million square kilometers of the African continent. Yet sleeping sickness has a nasty habit of springing back if expensive control efforts are relaxed.

When Aksoy arrived at Yale in the 1980s after earning her Ph.D. at Columbia University, a sleeping sickness epidemic had raged in Africa for over a decade. Eager to study the trypanosome parasite’s ever-shifting, vaccine-resistant surface coat, she joined the molecular biology laboratory of Dr. Frank Richards. Over time, she grew interested in how the parasite and fly interacted, eventually setting up a tsetse colony and spending over two decades studying it.

“The fly was such a black box it took us a long time to build the molecular foundation, then discover the genes and the biological nuts and bolts of the fly to be able to understand the interactions (with the trypanosome that infects it),” Aksoy recalled.

But the groundwork paid off. In 2014, she co-authored a landmark paper in Science reporting tsetse’s genome sequence, reported by the consortium she initiated and led comprised of small labs around the world, including Yale- affiliated bioinformaticians in Kenya and South Africa. The paper yielded clues about the fly’s sense of smell, how it recognizes pathogens and its ability to lactate.

You read that correctly: almost uniquely among insects, the tsetse fly undergoes a pregnancy during which it nurtures a single intrauterine larva with milk. Through this milk, the mother fly passes to its offspring two bacterial species that have coevolved with the fly. One, Wigglesworthia, makes B vitamins that are absent from the fly’s vertebrate blood-specific diet. Eliminating Wigglesworthia with antibiotics stops the fly’s ability to lactate, halting reproduction.

Tsetse flies also need these bacteria to develop a functioning immune system. If the researchers eliminate them and then supplement the diet with yeast extract to replace missing bacterial nutrients, the fly can once again reproduce. But the offspring—called aposymbiotic, or lacking in symbiotic bacteria—aren’t the same. Their immune systems don’t develop properly, rendering them susceptible to try- panosome infection.

Normally, after ingesting trypanosomes with a blood meal, most flies can eliminate the parasites. But among adults lacking their maternal microbiota, over half wind up contracting trypanosome infections. The presence of the bacteria during pregnancy is somehow key to helping flies clear the infections when they emerge as adults. These aposymbiotic adults also wind up having their energy sapped; their fecundity also drops.

“If you look at the gut of an infected tsetse fly, it’s unbelievable. I’ve been doing this for a long time and it fascinates me every time—the gut is just packed with these parasites,” said Brian L. Weiss, Ph.D., a research scientist who has worked with Aksoy for 15 years. “And they’re competing for nutrients with the fly.”

Healthy flies fight off trypanosomes in part via a sleeve- like structure called the peritrophic matrix. The matrix lines the gut, forming a barrier that trypanosomes have to muscle through twice during their life cycle. Healthy flies produce a thicker matrix and are likelier to resist such manipulation. By contrast, the matrix of aposymbiotic flies is thinner, porous, even nonexistent. That could explain their susceptibility to trypanosome infection. Trypanosomes also facilitate their own journey through the tsetse vector by shedding surface proteins that trick the fly into temporarily producing a less robust matrix, the Aksoy lab discovered.

But it is the fly’s other commensal bacterial species, Sodalis, that might offer the most practical key to disease control. Sodalis lives in the gut, where it comes into contact with trypanosomes. Unlike Wigglesworthia, Sodalis can be cultured in the lab. Weiss is exploring the possibility of adding new genes to Sodalis that give it anti-trypanosome powers.

“You could get them to produce molecules that are directly toxic to the trypanosomes. Or maybe you could get them to produce proteins that would bind to the peritrophic matrix and make it stronger, and thus the trypanosomes would be less able to penetrate it,” Weiss said.

Reintroducing genetically modified Sodalis into the fly creates a so-called paratransgenic system, one in which the trypanosome can’t gain a foothold.

“Our goal is to use these paratransgenic systems to strengthen the gut barriers,” Aksoy said. Trypanosome-resistant paratransgenic female flies could be released into the wild, where they would presumably mate with their wild male counterparts. Thanks to lactation, the protective bacteria would pass from mother flies to new generations.

But the technique’s safety and efficacy have yet to be demonstrated. Fortunately, Aksoy’s lab has long collaborated with researchers in Kenya and Uganda, who are in an excellent position to carry out the fieldwork. A grant from the NIH’s Fogarty International Center allowed the lab

to build tsetse research capacity with the Kenyan Agricultural & Livestock Research Institute. More recently, Aksoy secured an NIH International Centers for Excellence in Research grant to study fly population genetics and natural microbiota associated with flies also in collaboration with Kenyan researchers, a crucial step in predicting where paratransgenic flies could most effectively be released. In addition to university partnerships, the lab has ties to Kenya’s International Centre of Insect Physiology and Ecology and Uganda’s National Livestock Resources Research Institute.

Along the way, Aksoy and her colleagues, including Dr. Adalgisa Caccone at Yale, have led workshops on research techniques and academic leadership skills for African Ph.D. students, postdocs and early-career researchers. Many African scientists have worked in Aksoy’s New Haven lab and are now junior faculty at institutions in their home countries.

“The big vision that we had in the field [is] to move these technologies into Africa, [where] they would be utilized by the African scientists who are more versatile with their own communities,” Aksoy said. “They will be the next-generation researchers in the tsetse field.” 

Read the latest edition of Yale Public Health magazine.

Submitted by Elisabeth Reitman on December 17, 2018